Inflammation is often induced by proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, IL-6, macrophage migration inhibitory factor (MIF), and other compounds. These proinflammatory cytokines are produced by several different cell types, most importantly immune cells (for example, monocytes, macrophages and neutrophils), but also non-immune cells such as fibroblasts, osteoblasts, smooth muscle cells, epithelial cells, and neurons. These proinflammatory cytokines contribute to various disorders during the early stages of an inflammatory cytokine cascade.
The early proinflammatory cytokines (e.g., TNF, IL-1, etc.) mediate inflammation, and induce the late release of high mobility group box 1 (HMGB1; also known as HMG-1 and HMG1), a protein that accumulates in serum and mediates delayed lethality and further induction of early proinflammatory cytokines. HMGB1 was first identified as the founding member of a family of DNA-binding proteins, termed high mobility group box (HMGB) proteins, which are critical for DNA structure and stability. It was identified as a ubiquitously-expressed nuclear protein that binds double-stranded DNA without sequence specificity. The HMGB1 molecule has three domains: two DNA binding motifs termed HMGB A and HMGB B boxes, and an acidic carboxyl terminus. The two HMGB boxes are highly conserved 80 amino acid, L-shaped domains. HMG boxes are also expressed in other transcription factors including the RNA polymerase I transcription factor human upstream-binding factor and lymphoid-specific factor.
HMGB1 has been implicated as a cytokine mediator of delayed lethality in endotoxemia (Andersson, U., et al., J. Exp. Med. 192(4):565-570 (2000)). That work demonstrated that bacterial endotoxin (lipopolysaccharide (LPS)) activates monocytes/macrophages to release HMGB1 as a late response to activation, resulting in elevated serum HMGB1 levels that are toxic. Antibodies against HMGB1 prevent lethality from endotoxin even when antibody administration is delayed until after the early cytokine response. Like other proinflammatory cytokines, HMGB1 is a potent activator of monocytes. It has been demonstrated that intratracheal application of HMGB1 causes acute lung injury, and anti-HMGB1 antibodies protect against endotoxin-induced lung edema (Abraham, E., et al., J. Immunol. 165:2950-2954 (2000)). It has further been shown that serum HMGB1 levels are elevated in critically ill patients with sepsis or hemorrhagic shock, and levels are significantly higher in non-survivors as compared to survivors (U.S. Pat. No. 6,303,321). In vivo administration of HMGB1 has been shown to induce arthritis when injected into murine joints (Pullerits, R., et al., Arthritis Rheum. 481693-1700 (2003)).
HMGB1 has also been implicated as a ligand for RAGE, a multi-ligand receptor of the immunoglobulin superfamily. RAGE is expressed on endothelial cells, smooth muscle cells, monocytes, and nerves, and ligand interaction transduces signals through MAP kinase, P21 ras, and NF-κB. In addition, HMGB1 binds Toll-like receptor 2 (TLR2) and inhibition of this interaction can decrease or prevent inflammation (U.S. Published Application No. 20040053841). It has also been shown that receptor signal transduction of HMGB1 occurs in part through Toll-like receptor 4 (TLR4) (Park, J. S. et al., J. Biol. Chem. 279(9):7370-77 (2004)).
The delayed kinetics of HMGB1 appearance during endotoxema make it a potentially good therapeutic target, but little is known about the molecular basis of HMGB1 signaling and toxicity. Given the importance of HMGB1 in mediating inflammation, it would be useful to identify antibodies that bind HMGB1 and fragments thereof, for diagnostic and therapeutic purposes.